Fig. 3. PP1 blocks both SCF-mediated Kit and MAPK activation. In
A, H526 cells incubated in serum-free medium overnight
were either left unstimulated or stimulated for 5 min with 100 ng/ml of
SCF after a 30-min pretreatment with the indicated concentrations of
PP1. Kit was immunoprecipitated and the immunoprecipitate was
immunoblotted with antiphosphotyrosine and anti-Kit antibodies in
succession. The degree of Kit activation was calculated by densitometry
(ratio of pTyr:Kit signal) and normalized to the SCF-stimulated vehicle
control lane which was arbitrarily assigned a value of 100%.
Experiment shown is representative of three replicates. In
B, cells were treated as above, but after SCF treatment,
whole-cell lysates were made in SDS buffer. The lysates were
immunoblotted and stained successively with antiphospho-ERK and
antipan-ERK antibodies. The degree of activation was calculated as
above. Experiment shown is representative of three replicates.